1. Field of the Invention
The present invention relates to biopolymer production and in particular to a novel process for microbiologically producing short-chain polyhydroxyalkanoate (PHA) biopolymers using glycerol (glycerine) and levulinic acid as fermentation feedstocks. Furthermore the present invention relates to the production of short-chain PHA biopolymers having tunable monomer compositions and material properties using varying ratios of glycerol and levulinic acid.
2. Description of the Related Art
Increased environmental concern has prompted the search for “green” substitutes for many petroleum-based materials including transportation fuels, chemicals, and polymers. This search has lead to the development of novel uses for renewable and sustainable feedstocks as precursors for these bio-based products. Unfortunately, experience has shown that synthesis of bio-based materials generally involves higher overhead costs than petroleum-based products thereby hindering extensive use of bio-based materials in industrial applications.
Biopolymers are one class of chemicals that are currently receiving increased consideration as possible substitutes for petroleum-based polymers. However, successful replacement can only be realized if the biopolymers can be produced in adequate quantities from low-cost feedstocks and exhibit properties that are at least equivalent to their synthetic counterparts. Glycerol is one feedstock that is being assessed for new applications in both chemical and biological syntheses. It is created as a co-product in the base-catalyzed transesterification of triacylglycerols (TAGs) with short-chain alcohols, generally methanol in the biodiesel production process.
Historically, glycerol has been used in the drug, food, beverage, chemical and synthetic material industries. However, these applications normally require a high quality glycerol. The transesterification reaction involved in the biodiesel production process results in a crude glycerol component whose composition differs based on the type of TAG used, the efficiency of the transesterification reaction and the effectiveness of biodiesel and alcohol recovery. Yet, because of the rapid expansion of the biodiesel industry, glycerol is being produced at levels that have caused its value to drop to historic lows. While the price has recovered somewhat, the expected pace of future biodiesel production necessitates new uses for glycerol to help maintain some value.
Poly(hydroxyalkanoates) (PHA) represent a family of biodegradable bacterial polyesters that are synthesized as carbon and energy reserves by numerous bacterial species from many different carbon substrates (Huijberts, G. N. M., et al., Appl. Environ. Microbiol., 58: 536-544 (1992); Eggink, G., et al., Ind. Crops Products, 1: 157-163 (1993); de Smet, M-J., et al., J. Bacteriol., 154: 870-878 (1983); Ashby, R. D., and T. A. Foglia, Appl. Microbiol. Biotechnol., 49: 431-437 (1998); Solaiman, D. K. Y., et al., Curr. Microbiol., 44: 189-195 (2002). Synthesis generally occurs when a surplus of exogenous carbon is present and cellular growth is impeded by the lack of some other essential nutrient. PHA biopolymers are generally classified into 3 groups based on the length of their side-chains. Short-chain-length PHA (scl-PHA) polymers consist of 3-hydroxyalkanoic acids with monomeric repeat units of 3-5 carbons, medium-chain-length PHA (mcl-PHA) polymers are composed of monomeric repeat units that are 6-12 carbons in length, and long-chain-length PHA (lcl-PHA) polymers are comprised of monomeric repeat units ≧13 carbons. Because of their side-chain length and structural variability, PHA polymers exhibit a wide array of material properties from rigid thermoplastics to amorphous elastomers. Poly(3-hydroxybutyrate) (PHB), the simplest of the PHA biopolymers, was first discovered in 1926 and has been the best-characterized polymer within the PHA family (Lemoigne, M., Bull. Soc. Chem. Biol. (Paris), 8: 770-782 (1926)). It has been favorably compared to the petroleum-based polypropylene (PP) and polyethylene (PE) with respect to its environmental impact and its material properties. Life cycle analysis (LCA) showed that PHB was better than PP and PE in environmental impact by surpassing both PP and PE in its effects on abiotic and ozone depletion, global warming, human and ecotoxicity, acidification and eutrophication (Harding, K. G., et al., J. Biotechnol., 130: 57-66 (2007)). However, because of its highly crystalline nature, PHB is considered too rigid and brittle for widespread industrial application. To improve the material properties of these polymers, studies shifted to the production of copolymers to reduce the crystallinity of the material and to make it more ductile while at the same time decreasing the melting temperature to simplify processing conditions. Monomers such as 4-hydroxybutyric acid (4-HB), 3-hydroxyvaleric acid (3-HV), 4-hydroxyvaleric acid (4-HV), 3-hydroxyhexanoic acid (3-HHx) and 3-hydroxyoctanoic acid (3-HO) among others have been successfully copolymerized with 3-hydroxybutyric acid (3-HB), and in each case the material properties were improved over the PHB homopolymer (Kunioka, M., et al., Appl. Microbiol. Biotechnol., 30: 569-573 (1989); Holmes, P. A., et al., U.S. Pat. No. 4,477,654; Valentin, H. E., Appl. Microbiol. Biotechnol., 36: 507-514 (1992); Doi, Y., et al., Macromolecules, 28: 4822-4828 (1995); Budde, C. F., et al., Appl. Environ. Microbiol., 77: 2847-2854 (2011); Lianggi, Z., et al., Lett. Appl. Microbiol., 42: 344-349 (2006)).
Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB/V) is one of the PHA copolymers that has drawn interest. It is easy to synthesize and has been shown to possess a good balance of tensile strength and ductility and exhibits more desirable mechanical properties than PHB. Historically, PHB/V was synthesized using various combinations of simple sugars and either propionic acid or pentanoic (valeric) acid to vary the 3-HB:3-HV ratios within the copolymers (Pereira, S. M. F., et al., Polym. Engin. Sci., 48: 2051-2059 (2008); Yu, S. T., et al., Proc. Biochem., 40: 2729-2734 (2005)). Acetyl CoA has long been known to be the precursor molecule in the synthesis of PHB. It is synthesized through substrate level phosphorylation and can be utilized either as an energy and metabolite precursor in the tricarboxylic acid cycle (TCA) or can be used by the microorganism to produce PHA biopolymers. In Ralstonia eutropha, the most studied PHB producing bacterial strain, production begins through the condensation of one acetyl-CoA molecule with another to produce acetoacetyl-CoA by the action of the 3-ketothiolase enzyme. Acetoacetyl-CoA is then reduced to 3-hydroxybutyryl-CoA by acetoacetyl-CoA reductase and finally polymerized by the action of PHA synthase (Madison, L. L., and G. W. Huisman, Microbiol. Molecul. Biol. Rev., 63: 21-53 (1999)). When propionic acid is introduced as a co-substrate, propionyl-CoA is created which can condense with acetyl-CoA to produce the 5-carbon precursor, 3-ketovaleryl-CoA. This molecule is then further reduced to 3-hydroxyvaleryl-CoA and polymerized into the growing polymer chain (Doi, Y., et al., Macromolecules, 20: 2988-2991 (1987); Bramer, C. O., and A. Steinbuchel, Microbiology, 147: 2203-2214 (2001)). When grown in the presence of pentanoic (valeric) acid, the acid must first be enzymatically converted to valeryl-CoA, and then converted to 3-hydroxyvaleryl-CoA before it can be incorporated into the growing polymer backbone (Page, W. J., et al., Appl. Environ. Microbiol., 58: 2866-2873 (1992); Yu, J., et al., J. Biobased Mat. Bioenergy, 3: 113-122 (2009)). In both cases, under appropriate fermentation conditions, the use of propionic acid and/or pentanoic (valeric) acid as co-substrates with simple fermentable sugars results in PHB/V copolymers.
Since raw material costs account for the majority of the overall production cost of these biopolymers, efforts have been made to produce PHA polymers from less expensive starting materials. Some of these efforts have included such raw materials as soy molasses, wheat-based co-products, and glycerol (Solaiman, D. K. Y., et al., Biotechnol. Lett., 28: 157-162 (2006); Koutinas, A. A., et al., Enz. Microb. Technol., 40: 1035-104 (2007); Xu, Y., et al., Proc. Biochem., 45: 153-163 (2010); Ashby, R. D., et al., J. Polym. Environ., 12: 105-112 (2004); Cavalheiro, J. M. B. T., et al., Proc. Biochem., 44: 509-515 (2009); Shrivastav, A., et al., Int. J. Biol. Macromol., 47: 283-287 (2010); Zhu, C., et al., Biotechnol. Prog., 26: 424-430 (2010); Mothes, G., et al., Eng. Life Sci., 7: 475-479 (2007); Kawata, Y., et al., Biosci. Biotechnol. Biochem., 74: 175-177 (2010)). Under the conditions employed in each of these studies, PHB homopolymers were synthesized; however, in combination with other renewable inexpensive carbon sources, the possibility exists that co-polymeric PHA can be produced that exhibit improved material properties over PHB at reduced cost.
Glycerol is an inexpensive, renewable co-product that has drawn interest as a substrate for PHA polymer synthesis. Glycerol is derived from the base-catalyzed transesterification of triacylglycerols (TAGs) in the production of biodiesel. Due to the large amounts of biodiesel being produced worldwide, glycerol is flooding the market and driving its value down to a few cents per pound, making it more attractive as a feedstock for a number of value-added bio-based materials including 1,3-propanediol (Mu et al., Biotechnol. Lett., 28: 1755-1759, 2006), propylene glycol (Dasari et al., Appl. Catal. A: General 281, 225-231, 2005), epichlorohydrin (Santacesaria et al., Ind. Eng. Chem. Res., 49, 964-970, 2010), 3-hydroxypropionic acid (Raj et al., Proc. Biochem., 43, 1440-1446, 2008), succinic acid (Scholten et al., Biotechnol. Lett., 31: 1947-1951, 2009) citric acid (Papanikolaou and Aggelis, Lipid Technol., 21: 83-87, 2009), acrylic acid (Witsuthammakul and Sooknoi, Appl. Catal. A: General, 413-414, 109-116, 2012) single cell oils (Papanikolaou and Aggelis, supra), and glycolipid bio surfactants (sophorolipids) (Ashby et al 2005a), among others.
Levulinic acid synthesis is based on cellulosic waste materials. Because of this, economic projections indicate that levulinic acid production costs could fall to as low as $0.04-$0.10/lb depending on the scale of operation (Bozell et al., Res. Conserv. Recycl., 28, 227-239, 2000). Since levulinic acid is cheap and is a structural analogue of pentanoic acid, it has been assessed as a secondary substrate in PHA biosynthesis. Results have shown that elevated concentrations of levulinic acid tend to be toxic to many microorganisms, but success in the production of PHB/V biopolymers has been realized when low concentrations of levulinic acid were used in combination with other less harmful substrates. To this end, PHB/V copolymers have been produced from levulinic acid when used in conjunction with typical monosaccharide co-substrates such as glucose (Jang and Rogers, Biotechnol. Lett., 18, 219-224, 1996), fructose syrup (Chung et al., J. Microbiology, 39, 79-82, 2001), xylose (Keenan et al., Biotechnol. Prog., 20, 1697-1704, 2004) and gluconic acid (Kim et al., J. Microbiol., 47, 651-656, 2009) however; the maximum 3-HV content realized in these studies was 86 mol % regardless of the bacterial strain used. Other researchers successfully produced terpolyesters containing 3-HB, 3-HV and 4-HV from levulinic acid under controlled conditions (Gorenflo et al., Biomacromolecules, 2, 45-57, 2001, Yu et al., J. Biobased Mat. Bioenergy, 3, 113-122, 2009).
Thus there is a need to make PHA biopolymers more economical and impart additional value to glycerol. Glycerol and levulinic acid were used as low-cost fermentative co-substrates in the synthesis of PHB/V copolymers with wide-ranging 3-HB:3-HV ratios. While various methods for making biodegradable polyesters have been developed, there remains a need in the art for a method of making biocompatible and biodegradable polyesters with tunable properties from two large-volume, low-value coproducts. The present invention described below includes such methods which are different from related art methods for producing bacterial biodegradable polyesters.